All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
The field of metabolomics centers on the metabolic and biochemical events associated with a cellular or biological system. Metabolomics seeks to depict the steady-state physiological state of a cell or organism as well as dynamic responses of a cell or organism to genetic and environmental modulation. Metabolomic tools permit the detection of disease states, the monitoring of disease progression and patient response to therapy, the classification of patients based on biochemical profiles and the identification of targets for drug design.
An ideal metabolomic tool reveals the concentration of a particular molecular species of interest in a physiological environment. It allows one to visualize how its concentration varies across an organ, tissue or cell. It permits the detection of metabolite levels and the changes in metabolite levels in response to environmental stimuli, and allows these changes to be monitored in real time. Using various such tools should permit multiple analytes to be measured simultaneously, even analytes of different structural and functional classes.
No currently available technology addresses all these issues in a satisfactory manner. Non-aqueous fractionation is static, invasive, has no cellular resolution and is sensitive to artifacts, while spectroscopic methods such as NMRi (nuclear magnetic resonance imaging) and PET (positron emission tomography) provide dynamic data, but poor spatial resolution. The development of genetically encoded molecular sensors, which transduce an interaction of the target molecule with a recognition element into a macroscopic observable format, via allosteric regulation of one or more signaling elements, may facilitate some of the goals.
The most common reporter element employed in molecular sensors is a sterically separated donor-acceptor FRET pair of fluorescent proteins (GFP spectral variants or otherwise) (Fehr et al., 2002, Proc. Natl. Acad. Sci USA 99: 9846-51), although single fluorescent proteins (Doi and Yanagawa, 1999, FEBS Lett. 453: 305-7), enzymes (Guntas and Ostermeier, 2004, J. Mol. Biol. 336: 263-73) and bioluminescent molecules (Xu et al., 1999, Proc. Natl. Acad. Sci. USA 96: 151-56) have been used as well. FRET (fluorescence resonance energy transfer) refers to a quantum mechanical effect between a given pair of chromophores, consisting of a fluorescence donor and respective acceptor. Prerequisites for FRET are proximity of donor and acceptor, and overlap between the donor emission spectrum and the acceptor excitation spectrum. When the donor and acceptor are in close enough vicinity, the emission of the excited donor decreases while emission of the sensitized acceptor increases (see Fehr et al., 2004, Current Opinion in Plant Biology 7: 345-51, herein incorporated by reference in its entirety).
There are two general types of FRET used by biosensors: intermolecular and intramolecular (Truong and Ikura, 2001, Current Opinion in Structural Biology 11: 573-78, herein incorporated by reference). Intermolecular FRET occurs when the fluorescent donor and acceptor molecules are on different macromolecules. This form of FRET is difficult to quantitate because the stoichiometry of acceptors to donors can vary with transfection efficiencies and expression levels. Nevertheless, several examples of intermolecular FRET have been reported (for a review, see Truong and Ikura, 2001; and Wouters et al., 2001, TRENDS in Cell Biol. 11(5): 203-11).
Intramolecular FRET occurs when both the donor and acceptor molecules are fused to the same molecule. In this type of sensor, the binding domains must undergo conformational changes that are large enough to translate metabolite binding into a change in FRET. Ideally, sensor families should share similar three-dimensional structures but have different substrate specificities that cover a wide spectrum of substrates. Furthermore, ultra-high-affinity binding in the nanomolar range would facilitate the engineering of mutant “nanosensors” for different physiological detection ranges by site-directed mutagenesis.
Some molecular sensors additionally employ a conformational actuator (most commonly a peptide which binds to one conformational state of the recognition element), to magnify the allosteric effect upon and resulting output of the reporter element (i.e., Miyakawa et al., 1997, Nature 388: 882-87). The applicability of the method in the absence of a conformational actuator, and its generalizability to a variety of analytes, has recently been demonstrated using bacterial periplasmic binding proteins (PBPs) (Fehr et al., 2002; Fehr et al., 2003, J. Biol. Chem. 278: 19127-33; and Lager et al., 2003, FEBS Lett. 553: 85-9).
Members of the bacterial PBP superfamily recognize hundreds of substrates with high affinity (atto- to low micro-molar) and specificity (Tam and Saier, 1993, Microbiol. Rev. 57: 320-46). PBPs have been shown by a variety of experimental techniques to undergo a significant conformational change upon ligand binding. Fusion of individual sugar-binding PBPs with a pair of GFP variants has produced sensors for maltose, ribose and glucose (Fehr et al., 2002; Fehr et al., 2003; and Lager et al., 2003). Moreover, PBPs bind substrates with affinities in the nanomolar range (Fehr et al., 2004). Thus, PBPs satisfy many of the criteria important for an ideal biosensor. The sensors have been used to measure sugar uptake and homeostasis in living animal cells, and sub-cellular analyte levels have been determined using nuclear-targeted versions (Fehr et al., 2004, J. Fluoresc. 14: 603-9).
Intramolecular biosensors are typically designed by fusing donor and acceptor fluorescent molecules to the amino and carboxy terminal portions of the sensor domain, respectively, which undergo a venus flytrap-like closure of two lobes upon substrate binding (see, e.g., Fehr et al, 2002; Fehr et al., 2003; Lager et al., 2003; and Truong and Ikura, 2001). Bacterial PBPs comprise two globular domains and are convenient scaffolds for designing FRET sensors (Fehr et al., 2003). The binding site is located in the cleft between the domains, and upon binding, the two domains engulf the substrate and undergo a hinge-twist motion (Quiocho and Ledvina, 1996, Mol. Microbiol. 20: 17-25).
PBPs can be divided into two types based on different topological arrangements of the central β-sheets and position of the termini (Fukami-Kobayashi et al., 1999, J. Mol. Biol. 286: 279-290). Maltose binding protein (MBP) is a type II binding protein, with termini being located at the distal ends of the lobes relative to the hinge region. A comparison of the crystal structures of bound and unbound states shows that the hinge-twist motion brings the termini closer together. As would be expected in the case of maltose sensor, the decrease in distance upon maltose binding leads to increased FRET between attached chromophores (Fehr et al., 2002).
In GGBP (D-GalactoseD-Glucose Binding Protein) (a type I PBP), termini are located at the proximal ends of the two lobes (Fehr et al., 2004). Thus, because of the different chromophore positions, the substrate-induced hinge-twist motion is predicted to move the attached chromophores further apart, causing a decrease in FRET. Nevertheless, type I PBPs such as GGBP have also been used to construct efficient FRET biosensors containing terminally fused donor and acceptor fluorophores (Fehr et al., 2003).
The present inventors have now surprisingly found that fusion of fluorescent domains to internal positions of a ligand binding protein, even within the same lobe of a PBP sensor, facilitates the design of an efficient biosensor that demonstrates a similar ligand affinity and a substantially larger delta ratio than its terminally fused counterpart. This is counterintuitive in view of the general model for intramolecular FRET sensors, wherein the donor and acceptor molecules are fused to separate termini on separate lobes of the protein in order to maximize the change in orientation and/or distance of the donor and acceptor chromophores upon ligand binding.
The improved signal from these sensors can be ascribed to increased rigidity and thus reduced rotational averaging. The invention thus leads to an alternative approach, also disclosed herein, to improve sensors by using more rigidly conjugated reporters. To increase the rigidity and reduce rotational averaging, we deleted portions of the fusion proteins corresponding to residues not belonging to the core structure of the three contributing partners, i.e. omitting linker sequences at the fusion sites and deleting N- or C-terminal portions of either of the three modules. Consistent with the observations made for sensors using fusion of fluorescent domains to internal positions of a ligand binding protein, enhanced terminally fused sensors also showed much increased FRET ratio changes.